1. Field of the Invention
The present invention provides a method for obtaining thermostable enzymes. The present invention also provides variants of DNA polymerase I from Thermus aquaticus. The present invention further provides methods of identifying mutant DNA polymerases having enhanced catalytic activity. The present invention also provides polynucleotides, expression systems, and host cells encoding the mutant DNA polymerases. Still further, the present invention provides a method to carry out reverse transcriptase-polymerase chain reaction (RT-PCR) and kits to facilitate the same.
2. Discussion of the Background
Filamentous phage display is commonly used as a method to establish a link between a protein expressed as a fusion with a phage coat protein and its corresponding gene located within the phage particle (Marks et al., J. Biol. Chem. (1992) 267, 16007-16010). The use of filamentous phage particles as a chemical reagent provides further a strategy to create a complex between an enzyme, its gene and a substrate (Jestin et al., Angew. Chem. Int. Ed. (1999) 38, 1124-1127). This substrate can be cross-linked on the surface of filamentous phage using the nucleophilic properties of coat proteins. If the enzyme is active, conversion of the substrate to the product yields a phage particle cross-linked with the product, which can be captured by affinity chromatography (Jestin et al., Angew. Chem. Int. Ed. (1999) 38, 1124-1127)
Several similar approaches based on product formation for the isolation of genes encoding enzymes using phage display have been described in the literature for various enzymes (Fastrez et al., (2002) In: Brackmann, S. and Johnsson, K. eds., Directed Molecular Evolution of Proteins (Wiley VCH, Weinheim), pp 79-110). These in vitro selections of proteins for catalytic activity are well suited for use with large repertoires of about 108 proteins or more. Several libraries of enzyme variants on phage have been constructed and catalytically active proteins with wild type like activities have been isolated (Atwell & Wells (1999) Proc. Natl. Acad. Sci. USA 96, 9497-9502; Heinis et al. (2001) Prot. Eng. 14, 1043-1052; Ponsard et al. (2001) Chembiochem. 2, 253-259; Ting et al. (2001) Biopol. 60, 220-228.). Mutants with different substrate specificities have been also obtained (Xia et al. (2002) Proc. Natl. Acad. Sci. USA 99, 6597-6602.). In these studies, the fraction of active variants in the libraries can be large and it remains unclear how rare an enzyme can be in the initial protein library so as to be selected after iterative selection cycles. Accordingly, there remains a critical need for an efficient process for making and identifying thermostable enzymes possessing a desired catalytic activity (see discussion in Vichier-Guerre & Jestin, Biocat. & Biotransf. (2003) 21, 75-78).
Reverse transcriptases are enzymes that are present generally in certain animal viruses (i.e., retroviruses), which are used in vitro to make complementary DNA (cDNA) from an mRNA template. Practically, reverse transcriptases have engendered significant interest for their use in reverse transcriptase-polymerase chain reaction (RT-PCR). As such, these proteins lend themselves to be a model system for development of an efficient method of making thermostable enzymes having a desired activity.
RNA generally contains secondary structures and complex tertiary sections, accordingly it is highly desired that the RNA be copied in its entirety by reverse transcription to ensure that integrity of cDNA is maintained with high accuracy. However, due to the often complicated secondary and tertiary structures of RNA, the denaturation temperatures are generally about 90° C. and, as such, the reverse transcriptase must be capable of withstanding these extreme conditions while maintaining catalytic efficiency.
The classically utilized enzymes for RT-PCR have been isolated from the AMV (Avian myeloblastosis virus) or MMLV (Moloney murine leukemia virus); however, these enzymes suffer from a critical limitation in that they are not thermostable. In fact, the maximum temperature tolerated by most commercially available reverse transcriptases is about 70° C.
One common approach to overcome this limitation in the existing technology with the previously described polymerases has been the use of a protein chaperones in addition to the polymerase. However, this method leads to problems associated with environmental compatibility metal ion requirements, multi-stage procedures, and overall inconvenience. Accordingly, an alternative strategy has been to use thermostable reverse transcriptases. This approach makes it possible to perform multiple denaturation and reverse transcription cycles using only a single enzyme.
To this end, the DNA-dependent DNA polymerase I of Thermus aquaticus (i.e., Taq polymerase), is thermostable and has reverse transcriptase activity only in the presence of manganese. However, when the manganese ion concentration is maintained in the millimolar range the fidelity of the enzyme is affected. It has been suggested that the thermostable DNA-dependent DNA polymerase of Bacillus stearothermophilus has reverse transcriptase activity, even in absence of magnesium, but in this case it is necessary to add a thermostable DNA polymerase for the PCR.
Therefore, there remains a critical need for high efficiency, thermostable enzymes that are capable of catalyzing reverse transcription and subsequent DNA polymerization in “one-pot” RT-PCR. Accordingly, the present invention provides an isolated population of thermostable reverse transcriptases, which are active in absence of manganese, by directed evolution of the Stoffel fragment of the Taq polymerase.